The simian virus 40 (SV4O) tumor (T) antigen plays a crucial role in initiating and maintaining the replication of SV40 DNA in virus-infected monkey cells. This protein, along with the DNA and cellular factors involved, serves as a model to understand eukaryotic DNA replication. The work of many different labs has provided sole insights into the steps involved in this process but many details of this event are lacking. In particular, little is understood about the mechanism by which T antigen binds to and structurally distorts the origin. Likewise, we know little about how this protein unwinds the DNA at the origin and at replication forks, or about the nature and function of the molecular interactions between T antigen and the cellular factors engaged in DNA replication. Our approach has been to make numerous single-point substitution mutations in the origin-binding domain and elsewhere in the protein and to determine the effects of these mutations on various biochemical reactions that take place during initiation and propagation of DNA synthesis. In this way, we have identified amino acids that are likely to directly or indirectly participate in origin binding, nonspecific DNA binding, structural distortion of the origin, origin unwinding, oligomerization, and replication of DNA in vivo. By studying WT and various mutant proteins we propose to examine, in detail, the mechanisms by which T antigen prepares the origin for replication by melting and untwisting it, the way it unwinds DNA at the origin and at replication forks, how the interaction of T antigen with cellular proteins promotes DNA replication, and the role of T antigen in later stages of DNA replication. In this way, we hope to better understand the process by which viral and cell DNA replication takes place.